Bilirubin, a naturally occurring bile pigment generated during the physiological breakdown of heme, is a major component of bezoar bovis (dried ox gallstone), which has been used medicinally in the Far East for cancer chemoprevention. As epidemiological analyses support an inverse correlation between serum bilirubin levels and colorectal cancer mortality, and as bilirubin is normally present in the colonic lumen, the studies outlined in the current application are designed to test the novel hypothesis that bilirubin is the active ingredient of bezoar bovis and an inhibitor of intestinal tumorigenesis. The broad, long-term objectivesof the proposed research are to characterize the mechanism(s) underlying the chemopreventive effects of bilirubin and to explore potential therapeutic implications of these findings. The studies outlined will assess the efficacy of bilirubin in suppressing tumorigenesis in rodent models of colorectal cancer. Experiments will explore whether exogenously administered bilirubin prevents tumor development in the C57BL/6J-Min/+ (Min) strain of mouse, which possesses a fully penetrant dominant mutation in the Ape gene, and in rats treated with the carcinogen azoxymethane. The effect of increased endogenous levels of bilirubin on colon cancer formation will be analyzed in strains of congenitally jaundiced rodents with unconjugated (e.g., Gunn rats) or conjugated (TR- rats) hyperbilirubinemia. Finally, the impact of bilirubin treatment on intestinal inducible nitric oxide synthase (iNOS) and NADPH oxidase expression and activity will be assessed in the above rodent models. The results of these studies will hopefully provide a more comprehensive understanding of the role that bilirubin plays in the regulation of tumor development and growth. Since bilirubin is generally innocuous in adults, even at concentrations exceeding ten times normal, it is further anticipated that the results of these investigations will lay the foundation for potential new and effective therapies for the chemoprevention of colorectal cancer.